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The most reactive amide as a transition state mimic for cis-trans interconversion. Anthony J. Kirby J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja511460a • Publication Date (Web): 22 Dec 2014 Downloaded from http://pubs.acs.org on December 27, 2014
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The most reactive amide as a transition state mimic for cistrans interconversion.
Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:
Journal of the American Chemical Society ja-2014-11460a.R1 Article 08-Dec-2014 Komarov, Igor; Taras Shevchenko National University of Kyiv, Institute of High Technologies Yanik, Stanislav; Taras Shevchenko National University of Kyiv, Chemistry Ishchenko, Aleksandr; The Institute of High Technologies, Davies, John; University of Cambridge,, Goodman, Jonathan; University of Cambridge, Department of Chemistry Kirby, Anthony; University of Cambridge, Chemistry
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The most reactive amide as a transition state mimic for cis-trans interconversion. Igor V. Komarov*,1, Stanislav Yanik 2,3, Aleksandr Yu. Ishchenko 1,4, John E. Davies 5, Jonathan M. Goodman 5 and Anthony J. Kirby*,5 1
Institute of High Technologies and
2
Chemistry Department, Taras Shevchenko National
University of Kyiv, Volodymyrska 60 and 64, 01601 Kyiv (Ukraine). 3 Deceased. 4
Enamine Ltd., Aleksandra Matrosova 23, 01103 Kyiv (Ukraine)
5
University Chemical Laboratory, Cambridge CB2 1EW (UK)
ABSTRACT: 1-Azatricyclo[3.3.1.13,7]decan-2-one (3), the parent compound of a rare class of 90°-twisted amides, has finally been synthesized, using an unprecedented transformation. These compounds are of special interest as transition state mimics for the enzyme-catalysed cis-trans rotamer interconversion of amides involved in peptide and protein folding and function. The stabilization of the amide group in its high energy, perpendicular conformation common to both systems is shown for the rigid tricyclic system to depend, as predicted by calculation, on its methyl group substitution pattern, making 3 by some way the most reactive known "amide".
There is a compelling body of evidence that the cis-trans rotamer interconversion of amide bonds in peptides and proteins plays a fundamental role in countless biochemical processes. In addition to its involvement as the rate-limiting step in protein folding,1-3 recent studies have also identified key roles for the cis-trans interconversion in protein function. For example, signal transduction in immune cells involves peptidyl-prolyl bond cis-trans isomerization (Fig. 1) in secondary messengers, catalyzed by rotamase enzymes.4
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Figure 1. Peptidyl-prolyl bond cis-trans interconversion.
Similar deformations of the amide bonds in peptide substrates could in principle contribute to the catalytic efficiency of other classes of enzymes, including proteases5-8 and oligosaccharyltransferase,9 as well as to self-activation of proteins during splicing.10,11 And cis-trans isomerization in the peptide backbone of anion channel, 5-hydroxytryptamine type 3 (5-HT3) receptor, is reported to trigger the opening of the channel upon neurotransmitter binding.12 We report the synthesis and a theoretical and experimental study of a molecule (3) which represents the closest transition state model for the cis-trans interconversion of simple amides. Our current understanding of the mechanisms of biochemical transformations of this sort relies in large part upon detailed knowledge of the structure and properties of reaction intermediates and transition states (TS) in simpler systems. Transition states correspond to lowest saddle points on reaction potential energy surfaces, and their structures typically involve unusual bond angles and partial bonds which cannot be mimicked using direct connectivity with first or second row elements.13,14 However, it is possible to design a stable molecule as a meaningful TS mimic if any conformational motion of the molecular fragments bearing the characteristic features of the TS would lead to a sharp overall potential energy increase. This is possible in rigid systems, where conformational motions are severely restricted. Of course a stable TS mimic can only offer a crude approximation of the corresponding short-lived TS,15 but the degree of the approximation can be assessed by high-level computational techniques. In 1998 we reported the synthesis of the “highly twisted amide” 1,16 an extreme case of a bridged lactam,17 with the N–C=O fragment embedded in the rigid 1-azaadamantane skeleton, as a potential TS mimic for the cis-trans rotamer interconversion of the amide bond. Subsequent computational studies by Morgan et al.18 came to the conclusion that “the use of amide 1 as a model of the TS to amide C–N bond rotation carries some risk.” Comparing the calculated enthalpies for the hydrolysis of 1 and its di-, mono-, and non-methyl-substituted derivatives revealed significant buttressing effects of the methyl substituents on the reactivity of 1, leading to “artificial” ACS Paragon Plus Environment
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stabilization of this TS mimic. More recently Tani and Stoltz19 reported on the parent system 4 of the known Pracejus 2-quinuclidone 5, which is similarly stabilized by methyl substitution.20 In the absence of this evidently substantial stabilization the free base 4 could not be isolated, though the tetrafluoroborate salt was obtained, and its crystal structure reported.19
Results and discussion We report the synthesis and spectroscopic characterisation of 2, the monomethyl derivative of 1, and of the parent system 3: together with single crystal X-ray diffraction structures for 2 and for the conjugate acid 3.HBF4 of the parent system. Detailed comparisons of chemical reactivity, consistent with the calculations of the Morgan group, make 3 the most reactive known amide.
O
N
N
1
O
N
2
+ NH
2
COO-
O
N
3
4
+ NH
2
1. MeOH
COO1
2. Sublimation 6
98%
O
7
N
O
5
1. MeOH 2 2. Sublimation 41%
Figure 2. The highly twisted amide "family”: compounds 1-5 and the preparation of 1 and 2.
Synthesis of twisted amides. Twisted amide 1 was prepared from the amino acid 6 (obtained in seven steps from the Kemp triacid 21): 6 is converted spontaneously to some 80% of 1 in solution in methanol under mild, neutral conditions.22 The monomethyl derivative 2 is also formed spontaneously on dissolving the corresponding monomethyl-substituted amino-acid 7 in methanol: but significantly (about 10-fold) more slowly. In both cases evaporation of the methanolic solution followed by sublimation provides pure twisted amide. However, the synthesis of 3 presented a major challenge. According to Morgan's calculations,18 the methyl alpha to the carboxyl group should exert the biggest stabilising effect. So it was no surprise to find that the amino-acid 9, lacking the α-methyl group, did not react when dissolved in methanol: or in dry CD3CN, in which both 6 and 7 cyclised
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faster than we could run their 1H-NMR solution spectra. Standard methods of amide formation also failed. The clue to the successful synthesis of the parent compound 3 came from the observation of a strong peak at m/e 151 during the GS-MS identification of the N-Boc protected amino acid 8 23 (molecular ion m/e 269, see the Supplementary Information, SI). Boc-protection is known to be thermally labile,24 suggesting that the m/e 151 peak could correspond to the twisted amide 3, formed by decomposition of 8 under the conditions of the gas chromatography (column temperature 210 ºC).
Figure 3. Preparation of 3.
The preparative synthesis of 3 was seriously hampered by the released water. Compounds 3 and 8 both prove to be highly volatile, and difficult to separate from water in the gas phase. Thus simple sublimation of 8 in vacuum at temperatures between 180-250 ºC leads consistently to 3 heavily contaminated with starting material 8 and the amino-acid 9, the hydrolysis product of the evidently very reactive twisted amide. After extensive experimentation, we found that vacuum sublimation at elevated temperature of 8 pre-absorbed on silica gel produces pure 3 in moderate (43%) yield. Under these conditions, the twisted amide product 3 presumably remains trapped on the silica surface while water evaporates off: compound 3 finally collecting on the condenser. (The method also works well for the preparation of twisted amides 1 and 2, from the corresponding N-Bocprotected amino-acids, in yields of 63% and 51%, respectively.) Full details of the synthesis, spectroscopic evidence and crystal structure determinations confirming the structural assignments appear in the SI.
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Structure and spectroscopic properties. Compound 2 is sufficiently stable in the absence of (other) nucleophiles for full characterization. Crystalline 3 is thermally moderately stable, but polymerises in solution within a few hours. Nevertheless, with care we could characterize compound 3 spectroscopically. We also prepared single crystals of both 2 and 3 for X-Ray study, by slow vacuum sublimation. The molecular structure of 2 is shown in in Figure 4; but the crystal structure of 3 – typically for an adamantane derivative 25 – appears to be severely disordered, and no solution has so far proved possible. The crystal structure of the conjugate acid 3.HBF4 was solved, and is included in Fig. 4. Selected structural and spectroscopic parameters for 90° twisted amides 1, 2 and 3 and 2-quinuclidone 4 are shown in the Table: bond lengths and angles are calculated where crystal structures are unavailable (for 3 and 4: this work): for the reference unconstrained amides Nmethyl-piperidone 10 and N,N-dimethylacetamide 11, and for the syn- and anti-TS for the cis-trans interconversion of 11.18 The key spectroscopic parameters for twisted amides 1-3 are almost identical – and differ substantially from those for the unconstrained amides. As previously described for compound 1, these properties indicate that twisted amides 2 and 3 lack the resonance interaction between the nN and π*C=O orbitals that defines a typical amide group. Consequences are the major downfield 13C(O) NMR shift compared to that of the unconstrained amides, the IR C=O stretching band above 1730 cm-1, and ready fragmentation of the molecular ion, with the loss of a CO molecule, in the mass spectra (EI ionization). The crystal structures of 2 and 3.HBF4 (Fig. 4) confirm that these compounds belong to the “most twisted amide” family, with twist angles close to 90 degrees. The close agreement of calculated with crystal structures where both are available (Table S.1.1 in the Supplementary Information) is strong evidence that our calculated geometry (Table) gives an accurate description of the parent compound 3.
Figure 4. Molecular structures of twisted amides 2 (left) and 3.H+. ACS Paragon Plus Environment
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Table. Selected structural parameters and spectroscopic properties for twisted amides 1-3 and 4, for Nmethyl-2-piperidinone 10 26; and for N,N-dimethylacetamide 11, including transition states for its cistrans interconversion.18 Compound
1
2
3a
4a
4b
10
11
11 TS syn
11 TS anti
90
90
Selected structural parameters Twist angle τ, deg
90.5
90.0
90.55
105.05
90.0
2.5
0
Σ bond angles at N
325.7
325.6
326.9
324.2
327.1 358.9 360
338.6
332.7
Σ bond angles at C=O
359.9
359.9
360
359.8
360
359.9 360
360
360
C-N bond length, Å
1.475
1.448
1.450
1.443
1.433 1.375 1.38
1.45
1.46
C=O bond length, Å
1.196
1.201
1.203
1.203
1.183 1.232 1.23
1.21
1.21
Selected spectroscopic data c δ13C of C=O d
199.5
199.8
198.6
165
171
–
–
IR νmax of C=O, cm-1
1732
1732
1733
1653
1645
–
–
EI-MS 100% peak
(M-CO) +
10. CH2=N+Me
11. MeCO+
Notes. Data from X-ray structures (1, 2) or calculation: this work (3, 4) and Morgan (10, 11).18 See the text. a Calculations using 6-31++G**/M06-2X This work (for details see SI). b
Calculations at the RHF/6-31G* level: Greenberg, A.; Venanzi, C. A. J. Am. Chem. Soc. 1993,
116, 6951. c Data from this work (1 – 3) and standard literature sources. d 13C-NMR spectra run in CD3CN. Structurally, all three twisted amides 1-3 correspond closely to the syn-TS structure calculated for the cis-trans rotamer interconversion of N,N-dimethylacetamide 11 by Morgan et al.18 (Table) and by other authors.7 Planar carbonyl groups, highly pyramidalized nitrogen atoms and substantially elongated C–N and shortened C=O bonds are the key structural features characterising these compounds. The addition of methyl groups has no appreciable influence on the spectroscopic and structural parameters of the twisted amide fragment. However, the chemical reactivity of the three twisted amides 1-3 depends significantly on the number of bridgehead methyl groups. Comparisons with the 2-quinuclidone system 4 remain of interest,27 but we chose 3 as our ultimate synthetic target because the six-membered rings in the 2,2,2-system are restricted to the energetically less favourable boat conformation, with the majority of its substituent groups conformationally eclipsed. ACS Paragon Plus Environment
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The calculated twist angle τ of 105.5° shown for 4 in the Table and the "barrel-distortion" involved (Fig. S1 of the SI) applies to a minimum in our new high-level gas phase calculation: more significant is the related shallow dependence of the energy of the system on conformation change, which makes the 2-quinuclidone less rigid than the azaadamantanone system.
Chemical behavior. The chemistry of the monomethyl-substituted twisted amide 2 is closely similar to that of the trimethyl homologue 1. It reacts with nucleophiles slightly faster than 1, also at rates inconceivable for normal, unconstrained amides. However, the parent compound 3 is significantly more reactive towards nucleophiles than either 1 or 2. It reacts readily with trace amounts of water in "dry" solvents, so that NMR spectra need to be run as fast as practical. For direct comparisons we use the conditions described previously by Stoltz19 for the hydrolysis of the conjugate acid of the parent 2-quinuclidone 4. The hydrolysis experiments for 3 and 3.HBF4 were run in duplicate, and the half-lives found to be reproducible within a few seconds. The half-life of 4.HBF4 (0.084M) in acetonitrile-D3 solution in the presence of 5 equivalents of water was reported to be 135 min. We prepared the analogous tetrafluoroborate salts of 1-3 by HBF4 neutralization of the free base in dry acetonitrile followed by precipitation of the salts with diethyl ether. The salts are relatively stable in dry solvents, but react with water at very different rates. Under the Stoltz conditions the half-transformation times of 1.HBF4, 2.HBF4 and 3.HBF4 are 53.6 min, 103.5 min and 8.4 min., respectively. The relative rates are close to those calculated by Morgan for the hydration of the twisted amides, rather than those for hydrolysis to the aminoacids.18 In fact only 3.HBF4 is hydrolysed cleanly to the corresponding amino-acid (9.HBF4). Compound 1.HBF4 is converted to the tricyclic protonated hemiaminal 12 described previously,22 while hydrolysis of 2.HBF4 produces an equilibrium mixture of three compounds in solution: the starting material 2.HBF4, the corresponding protonated tricyclic hemiaminal 13 and the amino acid 7.HBF4: in approximate ratios of 1:3:4 (Fig. 5).
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BF4 D N+
O
D2O
BF4
D N+
OD OD
12 BF4
D N+
O
+ ND
half-life 53.6 min
BF4
2
D2O
COOD
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+
BF4 D N+
OD OD
half-transformation time 103.5 min
13 BF4 D N+
O
+ ND
BF4
2
D2O
COOD
half-life 8.4 min
Figure 5. Hydrolysis of the HBF4 salts of 1-3 (0.086M solutions in CD3CN, 5 equivalents of D2O, 23 °C)
Under these same conditions (5 equivalents of water in CD3CN) the half-life of compound 3, as the free base, is 16.9 min, making 3 the most reactive twisted amide: the trimethyl compound 1 does not react at all, while the monomethyl derivative 2 is converted – after > 3 hours – to an equilibrium mixture with the zwitterionic amino acid (2:7 ≈ 1.3:1, half-transformation time 17 min). The reaction of 3 with water is different again. It involves the formation of an intermediate, to which we assign the dimeric structure 14 (based on 1H- and 13C-NMR data: see the SI). The formation of 14 (together with traces of the corresponding trimer 15, n = 3) was confirmed by quenching the mixture obtained from reaction in THF-d8 with LiAlH4. (Experimental details appear in the SI). LC-MS analysis of the quenching products confirmed the formation of trimeric amino alcohols formed from 15, n = 3 (with the terminal CO2– reduced to CH2OH but amide bonds intact): or (depending on the conditions of the quenching) 15, n = 3 fully reduced to triamine alcohol: together with the dimeric equivalents based on 14 as the main products. Dimer 14 is presumably formed via acylation by unreacted 3 of its primary hydrolysis product, the zwitterionic amino acid 9. (Higher oligomers 15, n > 2 would be formed similarly by acylation of the dimer, etc.).
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Remarkably, these oligomeric intermediates 14 and 15 are themselves hydrolyzed under the conditions, eventually, after ca. 6 h, giving 9 almost exclusively. Intriguingly, the compounds with the carboxyl groups reduced withstand the aqueous work-up of the quenching products, suggesting a specific role for the carboxyl group in the rapid hydrolysis of 14 and the higher oligomers. Efficient intramolecular catalysis of the hydrolysis of non-activated amides by properly positioned carboxyl groups is described in the literature.28,29
Conclusions Structural parameters of the twisted amide groups in the three 1-azaadamantanones 1-3 closely match those for the TS for a model amide cis-trans interconversion, as calculated at high levels of theory. Both theory and experiment identify the parent molecule 3 as a valid TS model. The 90°twisted conformation of the amide group in the rigid azaadamantanone systems 1-3 is stabilized further by bridgehead methyl substituents. Just as the TS for the 90°-twisted conformation of a peptide amide group in the preorganised binding site of a rotamase enzyme must be stabilized by contributions from remote amino-acid side chains.30
Methods Standard methods were used for the preparation, isolation, and analysis of all new compounds (for details and characterisation see the SI). Preparation of 3 by pyrolysis/sublimation of 8. The pyrolysis/sublimation was carried out in a 50 mL round-bottom flask attached to a condenser equipped with a central cold-finger. The N-Boc amino-acid 8 (0.5-1.5 mmol: for its preparation see the SI) was dissolved in dry methanol (6 mL) in the 50 mL round-bottom flask. Silica gel (Kieselgel Merck 60, 5-times the weight of the N-Boc amino acid) was added to the solution, the methanol evaporated on a rotary evaporator (water vacuum pump, bath temperature 40 °C), and the residue dried in vacuum (water vacuum pump, bath temperature 40 °C) for ~30 min. Then the flask was connected to the condenser and a vacuum oil pump (~0.5 mm Hg). Connection to the vacuum has to be done with great care, because the mixture may produce volatile dust. The flask was immersed completely (up to the upper rim) in an oil bath, and the bath heated. Sublimation of product started at 115 °C (bath temperature). The temperature of the bath was increased to 150 °C over 1 h, then the product 3 was collected in the sublimer over 4h, while maintaining a bath temperature of 150±5 °C.
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ASSOCIATED CONTENT
Supporting Information. Crystallographic information files, calculated geometries, details of syntheses and kinetic measurements: and Spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Authors
[email protected] [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS
Financial assistance from Enamine Ltd. (www.enamine.net) is gratefully acknowledged. We also thank Dr. V. Stepanenko for valuable advice and encouragement which helped to find the nonstandard solutions to the synthetic problems, and Vitaliy Bilenko for drawing our attention to the instructive EI mass-spectrum of compound 8. REFERENCES
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The most reactive amide as a transition state mimic for cis-trans interconversion. Anthony J. Kirby J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja511460a • Publication Date (Web): 22 Dec 2014 Downloaded from http://pubs.acs.org on December 27, 2014
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The most reactive amide as a transition state mimic for cistrans interconversion.
Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:
Journal of the American Chemical Society ja-2014-11460a.R1 Article 08-Dec-2014 Komarov, Igor; Taras Shevchenko National University of Kyiv, Institute of High Technologies Yanik, Stanislav; Taras Shevchenko National University of Kyiv, Chemistry Ishchenko, Aleksandr; The Institute of High Technologies, Davies, John; University of Cambridge,, Goodman, Jonathan; University of Cambridge, Department of Chemistry Kirby, Anthony; University of Cambridge, Chemistry
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The most reactive amide as a transition state mimic for cis-trans interconversion. Igor V. Komarov*,1, Stanislav Yanik 2,3, Aleksandr Yu. Ishchenko 1,4, John E. Davies 5, Jonathan M. Goodman 5 and Anthony J. Kirby*,5 1
Institute of High Technologies and
2
Chemistry Department, Taras Shevchenko National
University of Kyiv, Volodymyrska 60 and 64, 01601 Kyiv (Ukraine). 3 Deceased. 4
Enamine Ltd., Aleksandra Matrosova 23, 01103 Kyiv (Ukraine)
5
University Chemical Laboratory, Cambridge CB2 1EW (UK)
ABSTRACT: 1-Azatricyclo[3.3.1.13,7]decan-2-one (3), the parent compound of a rare class of 90°-twisted amides, has finally been synthesized, using an unprecedented transformation. These compounds are of special interest as transition state mimics for the enzyme-catalysed cis-trans rotamer interconversion of amides involved in peptide and protein folding and function. The stabilization of the amide group in its high energy, perpendicular conformation common to both systems is shown for the rigid tricyclic system to depend, as predicted by calculation, on its methyl group substitution pattern, making 3 by some way the most reactive known "amide".
There is a compelling body of evidence that the cis-trans rotamer interconversion of amide bonds in peptides and proteins plays a fundamental role in countless biochemical processes. In addition to its involvement as the rate-limiting step in protein folding,1-3 recent studies have also identified key roles for the cis-trans interconversion in protein function. For example, signal transduction in immune cells involves peptidyl-prolyl bond cis-trans isomerization (Fig. 1) in secondary messengers, catalyzed by rotamase enzymes.4
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Figure 1. Peptidyl-prolyl bond cis-trans interconversion.
Similar deformations of the amide bonds in peptide substrates could in principle contribute to the catalytic efficiency of other classes of enzymes, including proteases5-8 and oligosaccharyltransferase,9 as well as to self-activation of proteins during splicing.10,11 And cis-trans isomerization in the peptide backbone of anion channel, 5-hydroxytryptamine type 3 (5-HT3) receptor, is reported to trigger the opening of the channel upon neurotransmitter binding.12 We report the synthesis and a theoretical and experimental study of a molecule (3) which represents the closest transition state model for the cis-trans interconversion of simple amides. Our current understanding of the mechanisms of biochemical transformations of this sort relies in large part upon detailed knowledge of the structure and properties of reaction intermediates and transition states (TS) in simpler systems. Transition states correspond to lowest saddle points on reaction potential energy surfaces, and their structures typically involve unusual bond angles and partial bonds which cannot be mimicked using direct connectivity with first or second row elements.13,14 However, it is possible to design a stable molecule as a meaningful TS mimic if any conformational motion of the molecular fragments bearing the characteristic features of the TS would lead to a sharp overall potential energy increase. This is possible in rigid systems, where conformational motions are severely restricted. Of course a stable TS mimic can only offer a crude approximation of the corresponding short-lived TS,15 but the degree of the approximation can be assessed by high-level computational techniques. In 1998 we reported the synthesis of the “highly twisted amide” 1,16 an extreme case of a bridged lactam,17 with the N–C=O fragment embedded in the rigid 1-azaadamantane skeleton, as a potential TS mimic for the cis-trans rotamer interconversion of the amide bond. Subsequent computational studies by Morgan et al.18 came to the conclusion that “the use of amide 1 as a model of the TS to amide C–N bond rotation carries some risk.” Comparing the calculated enthalpies for the hydrolysis of 1 and its di-, mono-, and non-methyl-substituted derivatives revealed significant buttressing effects of the methyl substituents on the reactivity of 1, leading to “artificial” ACS Paragon Plus Environment
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stabilization of this TS mimic. More recently Tani and Stoltz19 reported on the parent system 4 of the known Pracejus 2-quinuclidone 5, which is similarly stabilized by methyl substitution.20 In the absence of this evidently substantial stabilization the free base 4 could not be isolated, though the tetrafluoroborate salt was obtained, and its crystal structure reported.19
Results and discussion We report the synthesis and spectroscopic characterisation of 2, the monomethyl derivative of 1, and of the parent system 3: together with single crystal X-ray diffraction structures for 2 and for the conjugate acid 3.HBF4 of the parent system. Detailed comparisons of chemical reactivity, consistent with the calculations of the Morgan group, make 3 the most reactive known amide.
O
N
N
1
O
N
2
+ NH
2
COO-
O
N
3
4
+ NH
2
1. MeOH
COO1
2. Sublimation 6
98%
O
7
N
O
5
1. MeOH 2 2. Sublimation 41%
Figure 2. The highly twisted amide "family”: compounds 1-5 and the preparation of 1 and 2.
Synthesis of twisted amides. Twisted amide 1 was prepared from the amino acid 6 (obtained in seven steps from the Kemp triacid 21): 6 is converted spontaneously to some 80% of 1 in solution in methanol under mild, neutral conditions.22 The monomethyl derivative 2 is also formed spontaneously on dissolving the corresponding monomethyl-substituted amino-acid 7 in methanol: but significantly (about 10-fold) more slowly. In both cases evaporation of the methanolic solution followed by sublimation provides pure twisted amide. However, the synthesis of 3 presented a major challenge. According to Morgan's calculations,18 the methyl alpha to the carboxyl group should exert the biggest stabilising effect. So it was no surprise to find that the amino-acid 9, lacking the α-methyl group, did not react when dissolved in methanol: or in dry CD3CN, in which both 6 and 7 cyclised
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faster than we could run their 1H-NMR solution spectra. Standard methods of amide formation also failed. The clue to the successful synthesis of the parent compound 3 came from the observation of a strong peak at m/e 151 during the GS-MS identification of the N-Boc protected amino acid 8 23 (molecular ion m/e 269, see the Supplementary Information, SI). Boc-protection is known to be thermally labile,24 suggesting that the m/e 151 peak could correspond to the twisted amide 3, formed by decomposition of 8 under the conditions of the gas chromatography (column temperature 210 ºC).
Figure 3. Preparation of 3.
The preparative synthesis of 3 was seriously hampered by the released water. Compounds 3 and 8 both prove to be highly volatile, and difficult to separate from water in the gas phase. Thus simple sublimation of 8 in vacuum at temperatures between 180-250 ºC leads consistently to 3 heavily contaminated with starting material 8 and the amino-acid 9, the hydrolysis product of the evidently very reactive twisted amide. After extensive experimentation, we found that vacuum sublimation at elevated temperature of 8 pre-absorbed on silica gel produces pure 3 in moderate (43%) yield. Under these conditions, the twisted amide product 3 presumably remains trapped on the silica surface while water evaporates off: compound 3 finally collecting on the condenser. (The method also works well for the preparation of twisted amides 1 and 2, from the corresponding N-Bocprotected amino-acids, in yields of 63% and 51%, respectively.) Full details of the synthesis, spectroscopic evidence and crystal structure determinations confirming the structural assignments appear in the SI.
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Structure and spectroscopic properties. Compound 2 is sufficiently stable in the absence of (other) nucleophiles for full characterization. Crystalline 3 is thermally moderately stable, but polymerises in solution within a few hours. Nevertheless, with care we could characterize compound 3 spectroscopically. We also prepared single crystals of both 2 and 3 for X-Ray study, by slow vacuum sublimation. The molecular structure of 2 is shown in in Figure 4; but the crystal structure of 3 – typically for an adamantane derivative 25 – appears to be severely disordered, and no solution has so far proved possible. The crystal structure of the conjugate acid 3.HBF4 was solved, and is included in Fig. 4. Selected structural and spectroscopic parameters for 90° twisted amides 1, 2 and 3 and 2-quinuclidone 4 are shown in the Table: bond lengths and angles are calculated where crystal structures are unavailable (for 3 and 4: this work): for the reference unconstrained amides Nmethyl-piperidone 10 and N,N-dimethylacetamide 11, and for the syn- and anti-TS for the cis-trans interconversion of 11.18 The key spectroscopic parameters for twisted amides 1-3 are almost identical – and differ substantially from those for the unconstrained amides. As previously described for compound 1, these properties indicate that twisted amides 2 and 3 lack the resonance interaction between the nN and π*C=O orbitals that defines a typical amide group. Consequences are the major downfield 13C(O) NMR shift compared to that of the unconstrained amides, the IR C=O stretching band above 1730 cm-1, and ready fragmentation of the molecular ion, with the loss of a CO molecule, in the mass spectra (EI ionization). The crystal structures of 2 and 3.HBF4 (Fig. 4) confirm that these compounds belong to the “most twisted amide” family, with twist angles close to 90 degrees. The close agreement of calculated with crystal structures where both are available (Table S.1.1 in the Supplementary Information) is strong evidence that our calculated geometry (Table) gives an accurate description of the parent compound 3.
Figure 4. Molecular structures of twisted amides 2 (left) and 3.H+. ACS Paragon Plus Environment
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Table. Selected structural parameters and spectroscopic properties for twisted amides 1-3 and 4, for Nmethyl-2-piperidinone 10 26; and for N,N-dimethylacetamide 11, including transition states for its cistrans interconversion.18 Compound
1
2
3a
4a
4b
10
11
11 TS syn
11 TS anti
90
90
Selected structural parameters Twist angle τ, deg
90.5
90.0
90.55
105.05
90.0
2.5
0
Σ bond angles at N
325.7
325.6
326.9
324.2
327.1 358.9 360
338.6
332.7
Σ bond angles at C=O
359.9
359.9
360
359.8
360
359.9 360
360
360
C-N bond length, Å
1.475
1.448
1.450
1.443
1.433 1.375 1.38
1.45
1.46
C=O bond length, Å
1.196
1.201
1.203
1.203
1.183 1.232 1.23
1.21
1.21
Selected spectroscopic data c δ13C of C=O d
199.5
199.8
198.6
165
171
–
–
IR νmax of C=O, cm-1
1732
1732
1733
1653
1645
–
–
EI-MS 100% peak
(M-CO) +
10. CH2=N+Me
11. MeCO+
Notes. Data from X-ray structures (1, 2) or calculation: this work (3, 4) and Morgan (10, 11).18 See the text. a Calculations using 6-31++G**/M06-2X This work (for details see SI). b
Calculations at the RHF/6-31G* level: Greenberg, A.; Venanzi, C. A. J. Am. Chem. Soc. 1993,
116, 6951. c Data from this work (1 – 3) and standard literature sources. d 13C-NMR spectra run in CD3CN. Structurally, all three twisted amides 1-3 correspond closely to the syn-TS structure calculated for the cis-trans rotamer interconversion of N,N-dimethylacetamide 11 by Morgan et al.18 (Table) and by other authors.7 Planar carbonyl groups, highly pyramidalized nitrogen atoms and substantially elongated C–N and shortened C=O bonds are the key structural features characterising these compounds. The addition of methyl groups has no appreciable influence on the spectroscopic and structural parameters of the twisted amide fragment. However, the chemical reactivity of the three twisted amides 1-3 depends significantly on the number of bridgehead methyl groups. Comparisons with the 2-quinuclidone system 4 remain of interest,27 but we chose 3 as our ultimate synthetic target because the six-membered rings in the 2,2,2-system are restricted to the energetically less favourable boat conformation, with the majority of its substituent groups conformationally eclipsed. ACS Paragon Plus Environment
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The calculated twist angle τ of 105.5° shown for 4 in the Table and the "barrel-distortion" involved (Fig. S1 of the SI) applies to a minimum in our new high-level gas phase calculation: more significant is the related shallow dependence of the energy of the system on conformation change, which makes the 2-quinuclidone less rigid than the azaadamantanone system.
Chemical behavior. The chemistry of the monomethyl-substituted twisted amide 2 is closely similar to that of the trimethyl homologue 1. It reacts with nucleophiles slightly faster than 1, also at rates inconceivable for normal, unconstrained amides. However, the parent compound 3 is significantly more reactive towards nucleophiles than either 1 or 2. It reacts readily with trace amounts of water in "dry" solvents, so that NMR spectra need to be run as fast as practical. For direct comparisons we use the conditions described previously by Stoltz19 for the hydrolysis of the conjugate acid of the parent 2-quinuclidone 4. The hydrolysis experiments for 3 and 3.HBF4 were run in duplicate, and the half-lives found to be reproducible within a few seconds. The half-life of 4.HBF4 (0.084M) in acetonitrile-D3 solution in the presence of 5 equivalents of water was reported to be 135 min. We prepared the analogous tetrafluoroborate salts of 1-3 by HBF4 neutralization of the free base in dry acetonitrile followed by precipitation of the salts with diethyl ether. The salts are relatively stable in dry solvents, but react with water at very different rates. Under the Stoltz conditions the half-transformation times of 1.HBF4, 2.HBF4 and 3.HBF4 are 53.6 min, 103.5 min and 8.4 min., respectively. The relative rates are close to those calculated by Morgan for the hydration of the twisted amides, rather than those for hydrolysis to the aminoacids.18 In fact only 3.HBF4 is hydrolysed cleanly to the corresponding amino-acid (9.HBF4). Compound 1.HBF4 is converted to the tricyclic protonated hemiaminal 12 described previously,22 while hydrolysis of 2.HBF4 produces an equilibrium mixture of three compounds in solution: the starting material 2.HBF4, the corresponding protonated tricyclic hemiaminal 13 and the amino acid 7.HBF4: in approximate ratios of 1:3:4 (Fig. 5).
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BF4 D N+
O
D2O
BF4
D N+
OD OD
12 BF4
D N+
O
+ ND
half-life 53.6 min
BF4
2
D2O
COOD
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+
BF4 D N+
OD OD
half-transformation time 103.5 min
13 BF4 D N+
O
+ ND
BF4
2
D2O
COOD
half-life 8.4 min
Figure 5. Hydrolysis of the HBF4 salts of 1-3 (0.086M solutions in CD3CN, 5 equivalents of D2O, 23 °C)
Under these same conditions (5 equivalents of water in CD3CN) the half-life of compound 3, as the free base, is 16.9 min, making 3 the most reactive twisted amide: the trimethyl compound 1 does not react at all, while the monomethyl derivative 2 is converted – after > 3 hours – to an equilibrium mixture with the zwitterionic amino acid (2:7 ≈ 1.3:1, half-transformation time 17 min). The reaction of 3 with water is different again. It involves the formation of an intermediate, to which we assign the dimeric structure 14 (based on 1H- and 13C-NMR data: see the SI). The formation of 14 (together with traces of the corresponding trimer 15, n = 3) was confirmed by quenching the mixture obtained from reaction in THF-d8 with LiAlH4. (Experimental details appear in the SI). LC-MS analysis of the quenching products confirmed the formation of trimeric amino alcohols formed from 15, n = 3 (with the terminal CO2– reduced to CH2OH but amide bonds intact): or (depending on the conditions of the quenching) 15, n = 3 fully reduced to triamine alcohol: together with the dimeric equivalents based on 14 as the main products. Dimer 14 is presumably formed via acylation by unreacted 3 of its primary hydrolysis product, the zwitterionic amino acid 9. (Higher oligomers 15, n > 2 would be formed similarly by acylation of the dimer, etc.).
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Remarkably, these oligomeric intermediates 14 and 15 are themselves hydrolyzed under the conditions, eventually, after ca. 6 h, giving 9 almost exclusively. Intriguingly, the compounds with the carboxyl groups reduced withstand the aqueous work-up of the quenching products, suggesting a specific role for the carboxyl group in the rapid hydrolysis of 14 and the higher oligomers. Efficient intramolecular catalysis of the hydrolysis of non-activated amides by properly positioned carboxyl groups is described in the literature.28,29
Conclusions Structural parameters of the twisted amide groups in the three 1-azaadamantanones 1-3 closely match those for the TS for a model amide cis-trans interconversion, as calculated at high levels of theory. Both theory and experiment identify the parent molecule 3 as a valid TS model. The 90°twisted conformation of the amide group in the rigid azaadamantanone systems 1-3 is stabilized further by bridgehead methyl substituents. Just as the TS for the 90°-twisted conformation of a peptide amide group in the preorganised binding site of a rotamase enzyme must be stabilized by contributions from remote amino-acid side chains.30
Methods Standard methods were used for the preparation, isolation, and analysis of all new compounds (for details and characterisation see the SI). Preparation of 3 by pyrolysis/sublimation of 8. The pyrolysis/sublimation was carried out in a 50 mL round-bottom flask attached to a condenser equipped with a central cold-finger. The N-Boc amino-acid 8 (0.5-1.5 mmol: for its preparation see the SI) was dissolved in dry methanol (6 mL) in the 50 mL round-bottom flask. Silica gel (Kieselgel Merck 60, 5-times the weight of the N-Boc amino acid) was added to the solution, the methanol evaporated on a rotary evaporator (water vacuum pump, bath temperature 40 °C), and the residue dried in vacuum (water vacuum pump, bath temperature 40 °C) for ~30 min. Then the flask was connected to the condenser and a vacuum oil pump (~0.5 mm Hg). Connection to the vacuum has to be done with great care, because the mixture may produce volatile dust. The flask was immersed completely (up to the upper rim) in an oil bath, and the bath heated. Sublimation of product started at 115 °C (bath temperature). The temperature of the bath was increased to 150 °C over 1 h, then the product 3 was collected in the sublimer over 4h, while maintaining a bath temperature of 150±5 °C.
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ASSOCIATED CONTENT
Supporting Information. Crystallographic information files, calculated geometries, details of syntheses and kinetic measurements: and Spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Authors
[email protected] [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS
Financial assistance from Enamine Ltd. (www.enamine.net) is gratefully acknowledged. We also thank Dr. V. Stepanenko for valuable advice and encouragement which helped to find the nonstandard solutions to the synthetic problems, and Vitaliy Bilenko for drawing our attention to the instructive EI mass-spectrum of compound 8. REFERENCES
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